Multi-resistant Enterococcus faecium cause significant problems for both nosocomial infection treatment and control. Ampicillin is the therapy of choice for enterococcal strains that are susceptible to this agent. E. faecium are intrinsically resistant to low levels of ampicillin through expression of low affinity PBP5. Increased ampicillin resistance has been associated with both amino acid substitutions within PBPS and increased production of the protein. Ampicillin resistance has been tied to therapeutic failures and to increased risk of acquiring enterococcal gastrointestinal colonization. Work accomplished during the first three years of the project has identified the role of specific combinations of amino acid substitutions in PBP5-mediated resistance. We have also provided compelling evidence that increased production of PBP5 results from transcriptional activation of a bi-cistronic operon (psr-pbp5) by a diffusible activator specifically produced by highly resistant mutants. Finally, we have shown that E. faecium can acquire ampicillin resistance independently from PBP5 production by the activation of an alternate pathway of peptidoglycan cross-linking. This by-pass mechanism requires increased cellular carboxypeptidase activity to provide the substrate (tetrapeptide) of the L,D-transpeptidase that substitute for all the PBPs. In this competitive renewal, we will continue our analysis of structure-function relationships of PBPS, based on crystal structure studies of mutants already created. We will also isolate and characterize the transcriptional activator that binds upstream of psr, and explore more fully the involvement of another upstream gene, ftsWEfm, in ampicillin resistance expression. We will crystallize and characterize the structure of the novel L,D-transpeptidase to decipher the catalytic mechanism of this novel enzyme class. We will investigate the role of two putative carboxypeptidases in creating the tetrapeptide substrate required for L,D transpeptidation. Finally, we will determine which of the three Type A PBPs identified within the E. faecium genome cooperates with PBPS in synthesizing mature peptidoglycan. Successful completion of these experiments will yield a deeper understanding of the complex mechanisms by which E. faecium become resistant to ampicillin, a resistance phenotype that has important implications for the spread of enterococci within the hospital environment and the treatment of enterococcal infections in seriously ill patients.